HIGH THROUGH-PUT AUTOMATED DNA SEQUENCING

我们要求为更新加州理工学院成功的中央DNA测序核心设施的仪器提供资金。在过去的五年中，核心设施已经发展成为生物、化学、地质和环境工程部门正在进行的研究项目中不可或缺的贡献者。目前，我们每月为无预约用户制作约2500个场景。此外，核心设施还开展了两个由加州理工大学基因组资源中心协调的生产规模测序项目，除了许多BAC克隆外，还完成了整个微生物--嗜氧热杆菌--和人类16号染色体大片区域的测序。加州理工学院的所有实验室现在都依赖于自动化的、基于荧光的测序。面对日益增长的需求，为了继续保持我们已经实现的服务质量，我们需要三种类型的设备：一台ABI Prism 3700 DNA分析系统，新的聚合酶链式反应机器和几台小型计算机。我们选择了ABI Prism 3700，它基于一种新型的毛细管电泳系统，而不是平板凝胶，因为它有望实现最高的产量，以最低的成本、最少的劳动力和最高的质量成为目前可用的任何此类仪器。开发ABI3700的能力将需要聚合酶链式反应机器。需要计算机将新设备与现有的序列分析设备相连接，该设备将序列分发给用户。28个主要用户提供了他们将做的工作的摘要。将继续对单个cDNA、聚合酶链式反应产物、定点突变体等进行标准测序，这将为以下项目提供服务：RNA剪接、神经脊和/或肌肉发育的分子控制、果蝇身体节段的控制、非洲爪哇和酵母的细胞周期调控、几种生物的细胞命运和形态发生、拟南芥中与花发育有关的基因。我们希望，3700的巨大容量的可用性也将推动这里的实验室采用新的方法。例如，几个实验室(Anderson、Wold、Meyerowitz)正在开发策略，根据不同特定细胞类型和个体发育阶段的差异基因表达来识别cdna标记。另一个项目建议筛查人类线粒体中与年龄相关的突变。另一个维度是对整个微生物进行测序，并寻找具有有趣的已知路径的生物。这些后几个项目本身就可能涉及每月数千次的跑步。因此，我们希望保持我们作为一次性设施和高科技生产规模设施的能力，就像我们过去所做的那样。

We request funding for instrumentation to update a successful central DNA sequencing core facility at the California Institute of Technology. The Core Facility has developed over the last five years into an indispensable contributor to the ongoing research projects of Biology, Chemistry, Geology, and Environmental Engineering Divisions. Currently we are producing about 2500 sequences a month for walk-in users. In addition, the Core Facility has carried out two production scale sequencing projects coordinated by the Genome Resource Center at Caltech, completing the sequence of an entire micro-organism, Pyrobaculum aerophilum, and a large region of human chromosome 16, in addition to many BAC clones. All labs at Caltech now rely on automated, fluorescent-based sequencing. In order to continue the quality of service we have achieved in the face of increasing demand, we are requesting three types of equipment: an ABI Prism 3700 DNA Analysis system, new PCR machines, and several small computers. We have chosen the ABI Prism 3700, which is based on a novel capillary electrophoresis system rather than slab gels, because of its promise of the highest through-put, with the least labor at the lowest cost and with the highest quality of any such instrument currently available. The PCR machines will be needed to exploit the capacity of the ABI3700. The computers are needed to interface the new equipment with the existing Sequence Analysis Facility which distributes the sequence to the users. Twenty-eight major users provide abstracts of the work they will do. There will continue to be standard sequencing of single cDNAs, PCR products, site-directed mutants, etc. This will provide service to projects covering, among others, genes involved in RNA splicing, molecular control of neural crest and/or muscle development, in controlling body segment development in Drosophila, in cell cycle regulation in Xenopus and yeast, in cell fate and morphogenesis in several organisms, genes involved in floral development in Arabidopsis. Availability of the enormous capacity of the 3700 will, we hope, also drive new approaches in the laboratories here. For instance, several labs (Anderson, Wold, Meyerowitz) are developing strategies to identify cDNA markers based on differential gene expression for various specific cell types and individual stages of development. Another project proposes to screen age-related mutations in human mitochondria. Another dimension is sequencing entire microorganisms and panning for organisms with interesting known pathways. These latter projects could involve many thousands of runs per month in themselves. Thus, we hope to maintain our ability to function both as a walk-in facility and a high-tech production scale facility, as we have done in the past.